Prostanoids produced via the arachidonate cascade (prostaglandin (PG), thromboxane (TG), leukotriene (LT), etc.) are physiologically active substances which are essential in maintaining the homeostasis in vivo. Prostacyclin (PGI2), which is a prostanoid is synthesized in hemoendothelial cells under the action of PGI2 synthase on PGH2 having been formed from arachidonic acid by cyclooxygenase and prostaglandin H2 (PGH2) synthase.
It is known that the PGI2 has the following pharmacological effects:
(1) PGI2 binds to PGI2 receptor on membrane and activates adenyl cyclase to form cAMP, thereby exhibiting a platelet aggregation inhibitory effect and a vasodilating effect (Nakahata et al., Yakkyoku 50:1365–1373, 1999);
(2) PGI2 plays an important role in thrombus formation caused by hemoendothelial disorder (Murata, T. et al., Nature 388:678–682, 1997); and
(3) PGI2 is a major prostanoid inducing inflammatory edema (Murata, T. et al., Nature 388:678–682, 1997).
As described above, the biosynthesis pathway of PGI2 and the enzymes participating in its biosynthesis (arachidonate cascade) have been identified and thus the physiological activities of PGI2 have been clarified to a certain extent. However, the mechanism of regulating the PGI2 production in diseases still remains unknown.
Regarding the regulation of the PGI2 production, on the other hand, it has been suggested from the results of experiments with the use of spontaneously hypertensive rats (SHR), that an endogenous PGI2 production inhibitor may be present in SHR (Osanai, T. et al., Jpn. Circ. J. 54, 507–514, 1990; Falardeau, P. et al., Prostaglandins, 29, 621–628, 1985). Although this endogenous PGI2 production inhibitor has not yet been discussed sufficiently, the present inventors successfully isolated and purified from a SHR heart a PGI2 production inhibitory factor secreted by smooth muscle cells originating in SHR mesenteric artery. Further, they determined the structure of this factor and clarified that it is rat CF6 (hereinafter referred to as rCF6) which is one of the subunits of H+ ATP synthase Referential Example 1).
Mammalian H+ ATP synthase is present in the mitochondrial inner membrane and consists of at least 14 subunits. CF6, which is one of these subunits, consists of 76 amino acids. CF6 is synthesized as a peptide having 32 amino acids containing a mitochondrial transit sequence at the N-end (Higuchi, T. et al., Biochem. Biophys. Res. Commun. 178, 793–799, 1991).
The present inventors have established a method comprising expressing a chimeric protein from which rCF6 can be cut off by using factor Xa with the use of Escherichia coli as a host, and cutting off rCF6 from the chimeric protein followed by purification (Referential Example 2).
In addition, the present inventors have disclosed that the action point of the inhibition of the PGI2 production by CF6 resides in the PLA2 inhibition (Referential Example 3), in particular, in the cPLA2 inhibition (Referential Example 4) (Osanai, T. et al., J. Biol. Chem. 273, 31778–31778, 1998). However, the relationships between CF6, which is an intracellular peptide, and diseases have not been known so far. Moreover, it still remains unknown as to how CF6 acts on hemoendothelium in vivo, namely, whether or not CF6 is present in the blood.
On the other hand, it has been clarified the following facts with respect to the functions of cPLA2.
(1) cPLA2 is essentially required in the production of prostaglandin E2, leukotriene B4 and leukotriene C4 which are inflammatory mediators in the arachidonate cascade; and
(2) a mouse lacking this enzyme shows a small infarcted area after tentatively ligating the middle cerebral artery and scarcely suffers from edema or nerve defect in the brain (Bonventre JV, T. et al. Nature 390:622–625, 1997).
Based on these facts, it is estimated that cPLA2 inhibitors are usable as potent remedies for inflammation or remedies for brain infarction, etc. However, there has been found hitherto neither an efficacious remedy nor a method of screening a cPLA2 inhibitor on the basis of the effects of CF6.
As described above, the biosynthesis and physiological effects of PGI2, the physiological effects of cPLA2 in the arachidonate cascade and the relations among PGI2, cPLA2 and CF6 have been gradually clarified. However, causes of diseases, in which PGI2 located at the end of the arachidonate cascade and cPLA2 located at the initial point thereof participate, have not been sufficiently clarified yet.